**4.2 Analytical data and results interpretation**

By using the lab-investigations protocols, together with data processing and analysis using the chemometrics as described in previous sections, important information on the grape-based products from Romanian vineyards, either of organic and/ or conventional type. Synthetic data were presented in this sub-section, together with cross-references where details of the research may be found. However, at the moment of submission of this chapter, some experimental data are the subject of articles being drafted or under the review process, and may be consulted in the near future.

Phytochemical characterization of extracts prepared from grapes parts harvested from Romanian vineyards (organic and conventional management types) confirmed the variability described by the literature [91–93]. As examples, the type of vineyard management, the extraction solvent and/or method influenced the TPC, TFC, AA, pH, or conductivity of some prepared extracts, while for some others differences were not significant [3, 4, 19, 75].

**Table 2** presents some phytochemical parameters of grape skin, seeds and pulp (hydroalcoholic extracts obtained by room temperature maceration) of Feteasca Neagra variety of *Vitis vinifera L.,* harvested from both organic and conventional vineyards. One may observe that, for this grape variety, the total phenolic content, total flavonoids content and antioxidant activity in the extracts prepared from dry seeds is higher than in dry skin, and than in pulp. Also, once the vineyard type is considered, significant differences between the two types of culture management (organic/conventional) were recorded for TPC, TFC values of skins, seeds, and pulps, while for the AA, seeds extracts only showed significant differences. Some statistics are also provided in this table, with regards to grape varieties (a), and to phytochemical characteristics of extracts (b).

For the Pinot Noir variety, in the aqueous extracts prepared from organic grape skins a total flavonoids content of 0.317 ± 0.035 mg Quercetin/mL, almost triple than same extracts prepared from grapes originating from a conventional vineyard (0.109 ± 0.034 mg/mL), when the extraction method was classical maceration. For the case of ultrasound-assisted extraction, the TFC in organic grape skins aqueous extracts was over two-fold higher than the same kind of extracts but prepared from conventional cultivated grapes (recorded values were 0.297 ± 0.028 mg Quercetin/ mL, and respectively 0.139 ± 0.074 mg Quercetin/mL) [4]. The use of hydroalcoholic solvent showed similar behavior, in the sense that TFC was higher for samples from organic vineyards, than from conventional vineyard, but to a lower extent [3].


*a Significant difference (p* ≤ *0.05) among grapes' varieties. <sup>b</sup>*

*Significant difference (p* ≤ *0.05) between vineyard type, with regards to phytochemical characteristics of extracts (one-way ANOVA, Tukey test).*

#### **Table 2.**

*Phytochemical characteristics of Feteasca Neagra variety grapes parts (hydroalcoholic extracts).*

*Romanian Organic and Conventional Red Grapes Vineyards as Potential Sources… DOI: http://dx.doi.org/10.5772/intechopen.98972*

For two studied grape varieties a different behavior was found when the extraction procedure in water as solvent was applied. Thus, for Merlot (wine variety) and Muscat Hamburg (table grapes) aqueous extract, regardless the extraction method at room temperature (maceration and ultrasound-assisted), significant differences were recorded in the pH and conductivity measurements, when the vineyard type was considered. For the Merlot variety, pH and conductivity of the organic grapes skin extracts were always higher than for the conventional vineyard harvested samples, while for Muscat Hamburg variety an opposite variation was found for the pH (lower for organic originating samples extracts that for conventional ones), while no notable differences were found for conductivity values [4]. The explanation of these findings could be inferred from correlations with the specific treatments used in the vineyards, according to the management type of each culture [3, 4, 19, 75, 94], and further research is desirable.

For all studied grape varieties, regardless the solvent used in the initial step, the extracts prepared from dried seeds had higher values of TPC, TFC and AA than extracts prepared from dry skins and from grape pulp, regardless the vineyard type where the samples originated, and regardless the extraction method, if either maceration or ultrasound assisted, at room temperature [3, 4, 19, 40, 75].

For the hydroalcoholic extracts, while for the grape skins extracts TPC, TFC and AA had close values with regards to the vineyard type, if either organic or conventional, for the grape seeds' extracts, the experimental findings show significant differences between the organic and conventional samples, for these three phytochemical parameters, for the wine-type grapes (Feteasca Neagra, Merlot, Pinot Noir), while for the table grapes variety (Muscat Hamburg), the values were similar. The ANOVA algorithm, and the technique of multiple comparison applied on these measured values confirmed the differences between the antioxidants content (p < 0.05), and stated that TPC is the parameter the most influenced by the vineyard type, for both skins and seeds of studied grape varieties [3, 40].

A series of experiments were conducted aiming at evaluating whether the extraction procedures applied lead to obtaining samples with compounds that may have antimicrobial properties. Control samples without grapes extracts were tested for each set in the same conditions with the studied grapes extracts. Several bacteria strains were first isolated from ordinary environments, characterized and stored according to standardized procedures, and then used during the tests [3, 19, 40]. It was found that hydroalcoholic extracts prepared from grape skins originating from conventional type of cultures had a significant antibacterial activity against strains of *Lactococcus*, *Bacillus*, *Lactobacillus*, *Streptococcus*, *Leuconostoc*, *Micrococcus*, when compare to extracts obtained from the same varieties, but from grape skins originating from organic type of vine cultures. Another important experimental finding was that, when the hydroalcoholic solvent was used, the extracts of grape seeds from organic vineyards showed a broader spectrum of antibacterial activity than the seeds extracts from conventional vineyards grapes. Highest values of the antimicrobial activities, in seeds hydroalcoholic extracts, were found for the organic varieties of Merlot and Muscat Hamburg, and for the conventional Pinot Noir variety [3, 19, 40]. Antimicrobial activity data were subjected to statistical analysis, aiming at identifying correlations with phytochemical quantitative data [3, 40].

The mid-infrared spectroscopy with Fourier transformation (FTIR) has been used to obtain spectra of studied samples, in the wavenumbers range of 4000 cm−1 to 400 cm−1. **Figure 3** shows some examples of the spectra obtained for the native Romanian variety Feteasca Neagra, on hydroalcoholic extracts prepared from three anatomic parts of grapes harvested from organic, and respectively from conventional vineyards. As may be observed in this plot, measurements results are spectra with important similarities. Thus, all FTIR spectra showed strong peaks at 3275 cm−1,

**Figure 3.**

*Mid-infrared (FTIR) spectra recorded for grapes anatomic parts from organic (solid lines) and conventional (dashed lines) cultures of Feteasca Neagra vineyards (hydroalcoholic extracts).*

assigned to O-H stretching vibration, and in the range 1043–1055 cm−1, that may be assigned to C-O stretching, and to stretching vibrations of O-H and C-OH. Also, the peaks of 2979 cm−1 and around 2900 cm−1 could be assigned to asymmetric and symmetric stretching vibrations of -CH-, -CH2-. -CH3 from carbohydrates. The signal in the range of 1635–1643 cm−1 can be assigned to the aromatic C=C stretching vibrations which may correlate with the presence of anthocyanins, and also to C=O stretching vibration, while this finding may correlate with the presence of flavonoids like flavonols, flavons, isoflavones or flavanones. The peak recorded at 877 cm−1 was associated with the aromatic cycle C-H bending vibrations [4, 75, 77, 94]. Similar behavior was recorded for extracts of other grape varieties, provided by both organic and conventional vineyards, and are the subject of paper under review.

Unfortunately, information on some production parameters such as the irrigation level, crop yield, others, were not available for this study. Thus, further research will be considered, aiming at evaluating to what extent the recorded phytochemical data relate to the organic/conventional cultivation system only, and/ or to some specific agronomic practices.

*Romanian Organic and Conventional Red Grapes Vineyards as Potential Sources… DOI: http://dx.doi.org/10.5772/intechopen.98972*

As may be observed in **Figure 4**, similar spectra were obtained by using Raman spectroscopy, and the additional data processing and data analysis through chemometric techniques have been useful to extract further conclusions, and will be detailed below.

However, given the limited conclusions that may be extracted from the direct interpretation of the infrared and Raman spectra recorded for studied samples, chemometric methods have been applied considering the spectral data. Some results were published [94] and the following paragraphs will present some statistical analysis of samples indicated in **Table 3**, together with the additional information they could provide for the experimental findings. Codes indicated in this table correspond to those indicated in **Figure 4**. Multivariate analysis has been applied to FTIR and Raman spectral data recorded for hydroalcoholic extracts obtained from the four red grapes varieties indicated in the table, and for the three grapes parts studied - skin, seeds. and pulp.

#### **Figure 4.**

*Raman spectra recorded for grapes anatomic parts from organic (solid lines) and conventional (dashed lines) cultures of Feteasca Neagra vineyards (hydroalcoholic extracts).*


#### **Table 3.**

*Samples codes used in the chemometric analysis of spectral data.*

For the easiness of reading, conclusions extracted from statistical analysis were presented graphically in **Figure 5**. As may be observed, the figure shows information on the classification based on vineyard type, and the color and shape codes are explained in its caption. The work flow of the statistical analysis was as described in previous section.

A notable finding was that the decomposition of both FTIR and Raman spectral data through PCA revealed that with the first three principal components (PCs) a percentage higher than 90% of the total variability (the sum of percentage of variability explained by that PC and the preceding one) of the analyzed data was included. The PCA score plots showed that the investigated red grape varieties (*i.e.* skin extracts) overlapped (bootstrap ellipses) at different extent in all plots, and thus incomplete separations between varieties were noticed. However, it can be distinguished a separation between vineyard types (organic vs. conventional) for same grape variety (i.e., M-O vs. M-C, FN-O vs. FN-C, PN-O vs. PN-C and MH-O

**Figure 5.**

*Statistical classification of the red grapes hydroalcoholic extracts (skin/seeds/pulp), based on vineyard type (organic/conventional).*

*Romanian Organic and Conventional Red Grapes Vineyards as Potential Sources… DOI: http://dx.doi.org/10.5772/intechopen.98972*

vs. MH-C). The interpretation of the PCs loadings, for both FTIR and Raman spectral data, revealed the spectral regions/peaks that allow the differentiation between organic and conventional vineyards for same grape variety. Similar findings were recorded for the red grapes seeds and pulp extract studied.

Further analysis performed using Agglomerative Hierarchical Clustering (AHC) allowed a clear view of the similarities and differences between red grape parts extracts. For instance, AHC derived from grapes skins, FTIR data has grouped both organic and conventional extracts into two main classes/clusters (variance decomposition for the optimal classification: within-class 97.2%, between-classes 2.8%); at a lower dissimilarity level subclusters division allow a classification based on vineyard type (excepting PN-O), a differentiation was found for each grape variety between organic and conventional vineyards. From the classification obtained by using AHC based on Raman spectral data, organic and conventional extracts were similarly included into two main clusters (variance decomposition for the optimal classification: within-class 77.7%, between-classes 22.3%). Subclusters division based on Raman data shows notable differences between organic and conventional vineyards excepting Pinot Noir variety. The AHC algorithm applied on both FTIR and Raman data for seeds and pulp extracts lead also to grouping in two clusters, for both organic and conventional vineyards.

In the end, after the application of PCA on FTIR and Raman datasets, the first three principal components scores were retained for further analysis – classification and cross-validation through PC-DA. The result was, for all the three grape parts studied (skin, seeds, pulp) that all the extracts have been correctly classified through PC-DA, with only one exception (PN-O/FTIR data for skins).

For the case of the native Romanian variety Feteasca Neagra, and considering the vineyard management type only as criterion (conventional/organic), one may observe in **Figure 4** that application of AHC algorithm on FTIR data may provide a classification for all grape parts extracts (except FN-O/pulp) of the while the FTIR spectral data allow classification through, while application of the same algorithm on Raman data, a classification is possible only for seeds and pulp extracts. Another conclusion that may be extracted from **Figure 4**, is that once the PC-DA method is applied, a classification may be obtained while using both infrared and Raman spectroscopy datasets.

#### **5. Conclusions**

Romania is one of the major vine growers in the European Union, and in the same time, concerned with expanding the application of the principles of the circular economy in this field, with positive economic, social and environmental impacts on long term. The pedoclimatic conditions in the country offer the possibility of obtaining vine productions of an important variability, with qualitative and quantitative benefits. Subsequently, the composition of grape-based direct products (wine, food and beverages, others) and by-products (grape pomace, others) may vary, and thus leading to the desirable market variety. Extracting high-added value components from wastes in the vine-related industries may be a significant action in this context. Also, application of organic type of management to vineyards has the potential to significantly contribute to the sustainability in this field.

This chapter presents useful tools on how to characterize grape-based products extracts, and offers information on some cost-effective techniques suitable to collect, process and interpret experimental data. Thus, the information provided may contribute to taking informed decisions with regards to valorization of by-products generated in vine cultures.
